Mechanism Of Ubiquitin Transfer Research Articles

Abstract 2159: Deciphering the Mechanism of Ubiquitin Transfer by the Non-Canonical E2 Conjugating Enzyme, Ubc6

Abstract 2159: Deciphering the Mechanism of Ubiquitin Transfer by the Non-Canonical E2 Conjugating Enzyme, Ubc6

Molecular characterization of substrate-induced ubiquitin transfer by UBR7-PHD finger, a newly identified histone H2BK120 ubiquitin ligase.

Monoubiquitination of histone H2B at lysine 120 plays a vital role in active transcription and DNA damage response pathways. Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) has been recently identified as an H2BK120 monoubiquitin ligase. However, the molecular details of its ubiquitin transfer mechanism are not well understood. Here, we report that the plant homeodomain (PHD) finger of UBR7 is essential for its association with E2 UbcH6 and consequent ubiquitin transfer to its substrate histone H2B. We also identified the critical region of UbcH6 involved in this function and shown that the residues stretching from 114 to 125 of histone H2B C-terminal tail are sufficient for UBR7/UbcH6-mediated ubiquitin transfer. We also employed antibody-independent mass spectrometry to confirm UBR7-mediated ubiquitination of the H2B C-terminal tail. We demonstrated that the PHD finger of UBR7 forms a dimer and this dimerization is essential for ubiquitination of histone H2B. We mapped the critical residues involved in the dimerization and mutation of these residues that abrogate E3 ligase activity and are associated with cancer. Furthermore, we compared the mode of ubiquitin discharge from UbcH6 mediated by UBR7 and RING finger protein 20 (RNF20) through a thioester hydrolysis assay. Interestingly, binding of substrate H2B to UBR7 induces a conformational change in the PHD finger, which triggers ubiquitin transfer from UbcH6. However, the RNF20 RING finger alone is sufficient to promote the release of ubiquitin from UbcH6. Overall, the mechanism of ubiquitin transfer by the newly identified E3 ubiquitin ligase UBR7 is markedly different from that of RNF20.

Single-Domain Antibodies as Crystallization Chaperones to Enable Structure-Based Inhibitor Development for RBR E3 Ubiquitin Ligases

SummaryProtein ubiquitination plays a key role in the regulation of cellular processes, and misregulation of the ubiquitin system is linked to many diseases. So far, development of tool compounds that target enzymes of the ubiquitin system has been slow and only a few specific inhibitors are available. Here, we report the selection of single-domain antibodies (single-dAbs) based on a human scaffold that recognize the catalytic domain of HOIP, a subunit of the multi-component E3 LUBAC and member of the RBR family of E3 ligases. Some of these dAbs affect ligase activity and provide mechanistic insight into the ubiquitin transfer mechanism of different E2-conjugating enzymes. Furthermore, we show that the co-crystal structure of a HOIP RBR/dAb complex serves as a robust platform for soaking of ligands that target the active site cysteine of HOIP, thereby providing easy access to structure-based ligand design for this important class of E3 ligases.

The ubiquitin ligase SspH1 from Salmonella uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation

SspH/IpaH bacterial effector E3 ubiquitin (Ub) ligases, unrelated in sequence or structure to eukaryotic E3s, are utilized by a wide variety of Gram-negative bacteria during pathogenesis. These E3s function in a eukaryotic environment, utilize host cell E2 ubiquitin-conjugating enzymes of the Ube2D family, and target host proteins for ubiquitylation. Despite several crystal structures, details of Ube2D∼Ub binding and the mechanism of ubiquitin transfer are poorly understood. Here, we show that the catalytic E3 ligase domain of SspH1 can be divided into two subdomains: an N-terminal subdomain that harbors the active-site cysteine and a C-terminal subdomain containing the Ube2D∼Ub-binding site. SspH1 mutations designed to restrict subdomain motions show rapid formation of an E3∼Ub intermediate, but impaired Ub transfer to substrate. NMR experiments using paramagnetic spin labels reveal how SspH1 binds Ube2D∼Ub and targets the E2∼Ub active site. Unexpectedly, hydrogen/deuterium exchange MS shows that the E2∼Ub-binding region is dynamic but stabilized in the E3∼Ub intermediate. Our results support a model in which both subunits of an Ube2D∼Ub clamp onto a dynamic region of SspH1, promoting an E3 conformation poised for transthiolation. A conformational change is then required for Ub transfer from E3∼Ub to substrate.

Mechanism of ubiquitin transfer promoted by TRAF6

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) plays a vital role in immune signal transduction pathways by acting as a ubiquitin ligase (E3) for Lys63-linked polyubiquitin chain synthesis. However, the detailed mechanism by which the TRAF6 RING dimer promotes ubiquitin transfer was unknown. Through structural modeling and biochemical analysis, we here show that the TRAF6 RING dimer employs a concerted allosteric mechanism using both subunits of the TRAF6 dimer to promote ubiquitin (Ub) transfer. In particular, we reveal the importance of the C-terminal extension of the TRAF6 RING domain that mediates trans-interactions with the donor-Ub. By analyzing structures and models of E3s in complex with Ub-loaded ubiquitin-conjugating enzymes (E2s), we further highlight the roles of N-terminal and C-terminal extensions beyond the bona fide RING domains in promoting Ub transfer through engagement with a donor-Ub in cis and in trans, respectively.

Similar but Different: RBR E3 Ligases and their Domains that are Crucial for Function

Background: The E3 ubiquitin ligases can be subdivided into four distinct types (RING, HECT, U-box, and RBR type) based on their domain architecture and ubiquitin transfer mechanism. Recent structures of different RBR E3 ligases have been solved showing enzymes in their autoinhibited state. The only exception is HOIP/ HOIL-1L which was recently solved in its “active” conformation. This review discusses the structural and functional characteristics of three different members of the RBR E3 ubiquitin ligase family: Parkin, HOIP/HOIL-1L, and HHARI. Methods: Searches were performed using PubMed. Search term includes “RBR E3 Ligase”, “Parkin”, “HOIP/ HOIL-1L”, “HHARI”, “UbcH7”, and “E2”. In the end, 25 journal articles were selected as the foundation of this review. The structural coordinates of Parkin, HOIP, and HHARI were accessed from the PDB (www.rcsb.org) with the PDB IDs 4ZYN, 5EDV, and 4KBL, respectively. Summary: Currently, most solved RBR E3 ligase structures are only in their inactive forms, except for HOIP/ HOIL-1L, and these inactive forms provide valuable information on how these proteins are regulated in vivo. All the RBR E3 ligases have common domains, but their structures and functions are heavily dependent on their accessory domains, which serve as regulators that orchestrate certain ubiquitin chain syntheses and play a role in the autoinhibition of RBR E3 ligases. Although these domains are structurally different, they use distinct molecular interactions to achieve the same goal. While the regulation of most RBR E3 ligases has been extensively studied, more structural studies are required to further characterize the mechanism that these enzymes use to build different ubiquitin chains. Understanding the mechanisms underlying the formation of each type of ubiquitin chain could help elucidate their functions and related pathways.

Types of Ubiquitin Ligases

Ubiquitination is a post-translational modification of proteins involved in a variety of cellular processes. Ubiquitination requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases). This SnapShot highlights the main types of E3 ubiquitin ligases, which can be classified in three families depending on the presence of characteristic domains and on the mechanism of ubiquitin transfer to the substrate protein.

Identification and characterization of a ubiquitinconjugating enzyme UBE2A gene from lamprey.

Ubiquitin-conjugating enzymes (E2s) play an important role in the mechanism of ubiquitin transfer. Although in most species many of these enzymes share high sequence and structural conservation, their existence and functions in the lamprey remain unknown. In this study, we identified and characterized a ubiquitin-conjugating enzyme (UBE2A)-like gene in lamprey. The gene, designated as LaUBE2A, contained a 456-bp open reading frame encoding a 152-amino acid protein with a typical UBC domain. Real-time PCR assay showed that LaUBE2A was expressed in various tissues of the adult lamprey, with higher levels in the leukocytes and muscle and lower levels in the skin and liver. The high conservation in amino acid sequence between LaUBE2A and UBE2As from Homo sapiens, Mus musculus, Cavia porcellus, and Alligator sinensi implied that the function of LaUBE2A may be similar to that of UBE2A.

Chemical shift assignments for S. cerevisiae Ubc13.

The ubiquitination pathway controls several human cellular processes, most notably protein degradation. Ubiquitin, a small signaling protein, is activated by the E1 activating enzyme, transferred to an E2 conjugating enzyme, and then attached to a target substrate through a process that can be facilitated by an E3 ligase enzyme. The enzymatic mechanism of ubiquitin transfer from the E2 conjugating enzyme onto substrate is not clear. The highly conserved HPN motif in E2 catalytic domains is generally thought to help stabilize an oxyanion intermediate formed during ubiquitin transfer. However recent work suggests this motif is instead involved in a structural, non-enzymatic role. As a platform to better understand the E2 catalyzed ubiquitin transfer mechanism, we determined the chemical shift assignments of S. cerevisiae E2 enzyme Ubc13.

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.

Understanding E2 Mechanism using NMR

The ubiquitination pathway controls human cellular processes such as cell cycle division, DNA transcription and repair, and immune responses. In these processes, ubiquitin is covalently attached to a substrate protein by the combined efforts of an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ubiquitin ligase. We are concerned with investigating the catalytic mechanism of ubiquitin transfer onto the substrate protein. We specifically want to understand how the structure of Ubc13 helps with catalytic activity. The structure of yeast Ubc13, an E2 conjugating enzyme, has been solved using nuclear magnetic resonance (NMR). The study of the structure of Ubc13 compared to that of Ubc13 bound to ubiquitin in the active site will show the changes in the active site upon binding. Through the use of transition state mimics of ubiquitin conjugating reaction we will completely describe how Ubc13 stabilizes the ubiquitination conjugation pathway.

Exploring the RING-Catalyzed Ubiquitin Transfer Mechanism by MD and QM/MM Calculations

Ubiquitylation is a universal mechanism for controlling cellular functions. A large family of ubiquitin E3 ligases (E3) mediates Ubiquitin (Ub) modification. To facilitate Ub transfer, RING E3 ligases bind both the substrate and ubiquitin E2 conjugating enzyme (E2) linked to Ub via a thioester bond to form a catalytic complex. The mechanism of Ub transfer catalyzed by RING E3 remains elusive. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations, we characterized this catalytic mechanism in detail. The three-dimensional model of dimeric RING E3 ligase RNF4 RING, E2 ligase UbcH5A, Ub and the substrate SUMO2 shows close contact between the substrate and Ub transfer catalytic center. Deprotonation of the substrate lysine by D117 on UbcH5A occurs with almost no energy barrier as calculated by MD and QM/MM calculations. Then, the side chain of the activated lysine gets close to the thioester bond via a conformation change. The Ub transfer pathway begins with a nucleophilic addition that forms an oxyanion intermediate of a 4.23 kcal/mol energy barrier followed by nucleophilic elimination, resulting in a Ub modified substrate by a 5.65 kcal/mol energy barrier. These results provide insight into the mechanism of RING-catalyzed Ub transfer guiding the discovery of Ub system inhibitors.

Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred – E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions.

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine∼ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin's in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine-mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.

Decoding the SUMO signal

SUMO (small ubiquitin-like modifier) emerged from the shadow of the well-established ubiquitin some 15years ago when it was shown that a distinct conjugation pathway was responsible for SUMO modification. Since then it has been established that SUMO modifies over a thousand substrates and plays diverse roles in many important biological processes. Recognition of SUMO is mediated by short peptide sequences known as SIMs (SUMO-interaction motifs) that allow effector proteins to engage SUMO-modified substrates. Like ubiquitin, SUMO can form polymeric chains, and these chains can be recognized by proteins containing multiple SIMs. One protein that contains such a sequence of SIMs also contains a RING (really interesting new gene) domain that is the hallmark of a ubiquitin E3 ligase. This ubiquitin ligase known as RNF4 (RING finger protein 4) has the unique property that it can recognize SUMO-modified proteins and target them for ubiquitin-mediated proteolysis. Structural and biochemical analyses of RNF4 has shed light on the long sought after mechanism of ubiquitin transfer and illustrates how its RING domain primes the ubiquitin-loaded E2 for catalysis.

RING-between-RINGs-keeping the safety on loaded guns

The ‘RING‐between‐RING’‐type E3 ubiquitin ligase HOIP acts via a novel RING/HECT‐hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non‐covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is auto‐inhibited. This explains why assembly of either HOIP/HOIL‐1L or HOIP/SHARPIN is required to catalyse linear chain formation.

BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer

Certain RING ubiquitin ligases (E3s) dimerize to facilitate ubiquitin (Ub) transfer from ubiquitin-conjugating enzyme (E2) to substrate, but structural evidence on how this process promotes Ub transfer is lacking. Here we report the structure of the human dimeric RING domain from BIRC7 in complex with the E2 UbcH5B covalently linked to Ub (UbcH5B∼Ub). The structure reveals extensive noncovalent donor Ub interactions with UbcH5B and both subunits of the RING domain dimer that stabilize the globular body and C-terminal tail of Ub. Mutations that disrupt these noncovalent interactions or RING dimerization reduce UbcH5B∼Ub binding affinity and ubiquitination activity. Moreover, NMR analyses demonstrate that BIRC7 binding to UbcH5B∼Ub induces peak-shift perturbations in the donor Ub consistent with the crystallographically-observed Ub interactions. Our results provide structural insights into how dimeric RING E3s recruit E2∼Ub and optimize the donor Ub configuration for transfer.

Engineering a Ubiquitin Ligase Reveals Conformational Flexibility Required for Ubiquitin Transfer

Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.